Mechanical damage to the nervous system of mammals results in sometimes irreversible functional deficits. Most functional deficits associated with trauma to both the Peripheral Nervous System (PNS) or Central Nervous System (CNS) result from damage to the nerve fiber or axon, blocking the flow of nerve impulse traffic along the nerve fiber. This may be due to a physical discontinuity in the cable produced by axotomy. The blockage may also occur where the membrane no longer functions as an ionic fence, and/or becomes focally demyelinated [Honmou, O. and Young, W. (1995) Traumatic injury to the spinal axons (Waxman, S. G., Kocsis, J. D., Stys, P. K., Eds.): The Axon, New York: Oxford UP, pp 480-503; Maxwell, W. L. (1996): Histopathological changes at central nodes of ravier after stretch-injury, Microscopy Research and Technique, 34:522-535; Maxwell, W. L., Watt, C., Graham, D. I., Gennarelli, T. A. (1993): Ultrastructural evidence of axonal shearing as a result of lateral acceleration of the head in non-human primates, Acta Neuropathol, 86:136-144; Maxwell, W. L., Graham, D. I. (1997): Loss of axonal microtubules and neurofilaments after stretch-injury to guinea pig optic nerve fibers, J Neurotrauma, 14:603-614; Blight, A. R. (1993): Remyelination, Revascularization, and Recovery of Function in Experimental Spinal Cord Injury (Seil, F. J., Ed.): Advances in Neurobiology: Neural Injury and Regeneration, Vol. 59, New York, Raven Press, pp. 91-103]. In either case, functional deficits occur because of the break in nerve impulse conduction. Even the severe behavioral deficits associated with spinal cord injury is now understood to be largely due to the initial mechanical damage to white matter [Blight, A. R.: Morphometric analysis of a model of spinal cord injury in guinea pigs, with behavioral evidence of delayed secondary pathology, J. Neurolog. Sci., 103:156-171, 1991]. Delayed but progressive episodes of so-called “secondary injury” [Honmou and Young, W. (1995): Traumatic injury to the spinal axons (Waxman, S. G., Kocsis, J. D., Stys, P. K., Eds.): The Axon, New York: Oxford UP pp 480-503; Young, W. (1993): Secondary injury mechanisms in acute spinal cord injury, J. Emerg. Med, 11:13-22.] subsequently enlarge the lesion leading to the typical clinical picture of a cavitated contused spinal cord, and intractable behavioral loss.
In the mammal, transection of the axon leads to the irreversible loss of the distal nerve process segment by Wallerian degeneration, while the proximal segment may survive. In the PNS, function may be restored by the endogenous regeneration of proximal segments down fasciculation pathways provided by both connective tissue and Schwann cell “tubes” which may persist for variable amounts of time post injury (Risby, M. A. (1995): Regeneration of peripheral nervous system axons (Waxman, S. G., Kocsis, J. D., Stys, P. K., Eds.): The Axon Book, New York, The Oxford University Press, pp 553-578]. The level of the injury is critical to clinical fascicular repair however, as the rate of regeneration (about 1 mm/day) may not be sufficient to avoid loss of target tissues dependent on its innervation (such as motor units in striated muscle). In the CNS, distal segments of nerve fibers do not regenerate, and their loss produces nonfunctional “target” cells, which often require innervation to maintain their integrity. One ultimate strategy to enhance recovery from CNS injury is to induce or facilitate regeneration of white matter by various means.
In the clinic, acute spinal cord transection is rare while compressive/contusive mechanical damage is typical. In the PNS, transection, stretch injury as well as compression injury to nerve trunks are commonplace. However, severe, local, mechanical damage to any type of nerve fiber membrane may still initiate a process leading to axotomy and the irretrievable loss of distal segments. These events usually begin with a breakdown in the ability of the axolemma to separate and maintain critical differences in ions between the extracellular and intracellular compartments—in particular calcium.
The devastating effects of injury to the mammalian spinal cord are not immediate. Severe mechanical injury initiates a delayed destruction of spinal cord tissue producing a loss in nerve impulse conduction associated with a progressive local dissolution of nerve fibers (axons) [Honmou, O. and Young, W. (1995) The Axon (Waxman, S. G., et al., Eds.) pp. 480-529, Oxford University Press, New York; Griffin, J. W. et al. (1995) The Axon (Waxman, S. G., et al., Eds.) pp. 375-390, Oxford University Press, New York]. This loss of sensory and motor communication across the injury site can produce a permanent paralysis and loss of sensation in regions below the level of the spinal injury. Furthermore, it is clear the most damaging effects of progressive “secondary injury” [Young, W. (1993) J. Emerg. Med. 11:13-22] of spinal cord parenchyma relative to the loss of behavioral functioning is the effect it has on white matter. Localized mechanical, biochemical, and anoxic/ischemic injury to white matter may be sufficient to cause the failure of axolemmas to function as a bather or fence to the unregulated exchange of ions [Honmou, O. and Young, W. (1995) The Axon (Waxman, S. G., et al., Eds.) pp. 480-529, Oxford University Press, New York]. This in turn compromises both the structural integrity of this region of the nerve fiber and its ability to conduct impulses along the cable. For example, elevated intracellular Ca2+ induces depolymerization of microtubules and microfilaments producing a focal destruction of the cytoskeleton [Griffin, J. W. et al. (1995) The Axon (Waxman, S. G., et al., Eds.) pp. 375-390, Oxford University Press, New York; Maxwell, W. L., et al. (1995) J. Neurocytology 24:925-942; Maxwell, W. L., et al. J. Neurotrauma 16:273-284].
The unrestricted movement of Ca++ down its electrochemical gradient into the cell leads to a destruction of membranes and the cytosol, and is an initial key event in all mechanical injury to nerve fibers as well as other ischemic injuries such as head injury and stroke [Borgens, R. B., Jaffe, L. F., Cohen, M. J. (1980): Large and persistent electrical currents enter the transected spinal cord of the lamprey eel, Proc. Natl. Acad. Sci. U.S.A., 77:1209-1213; Borgens, R. B. (1988): Voltage gradients and ionic currents in injured and regenerating axons, Advances in Neurology, 47: 51-66; Maxwell, W. L. (1996): Histopathological changes at central nodes of ravier after stretch-injury, Microscopy Research and Technique, 34:522-535; Maxwell, W. L., Graham, D. I. (1997): Loss of axonal microtubules and neurofilaments after stretch-injury to guinea pig optic nerve fibers, J. Neurotrauma, 14:603-614; Maxwell, W. L., Watt, C., Graham, D. I., Gennarelli, T. A. (1993): Ultrastructural evidence of axonal shearing as a result of lateral acceleration of the head in non-human primates, Acta Neuropathol, 86:136-144; Honou and Young, 1995, Lee et al., 1999; Stys et. al., 1990]. Na+ enters the localized region of the membrane insult as well, depolarizing the membrane and facilitating the release of intracellular Ca++ stores [Carafoli, E., Crompton, M. (1976): Calcium ions and mitochondria (Duncan, C. J., Ed.): Symposium of the Society for Experimental Biology: Calcium and Biological Systems, Vol. 30, New York, Cambridge University Press, pp. 89-115; Borgens, R. B., Jaffe, L. F., Cohen, M. J. (1980): Large and persistent electrical currents enter the transected spinal cord of the lamprey eel, Proc. Natl. Acad. Sci. U.S.A., 77:1209-1213; 1988; Borgens, R. B. (1988): Voltage gradients and ionic currents in injured and regenerating axons, Advances in Neurology, 47: 51-66]. Potassium exodus also pushes the resting potential of the membrane towards the Nernst potential for K+ contributing to the localized region of inexcitability and blockage of nerve impulse conduction down the cable in even intact membranes. Thus, when K+ rushes down its electrochemical gradient out of the cell, the resultant elevated extracellular concentration contributes to localized conduction block [Honmou, O. and Young, W. (1995) The Axon (Waxman, S. G., et al., Eds.) pp. 480-529, Oxford University Press, New York; Shi, R. et al., (1997) Society for Neuroscience Abstracts, 108:16]. However it is the progressive chain reaction of events set in motion by Ca++ entry into the cell that initially leads to progressive dissolution of the axon—aided in later stages of the acute event by additional complex molecular processes such as the initiation of lipid peroxidation pathways and formation of “free radical” oxygen metabolites.
There are several classes of molecules that have already been shown to be able to seal cell membranes or to actually fuse membranes together [Nakajima, N., Ikada, Y. (1994): Fusogenic activity of various water-soluble polymers, J. Biomaterials Sci., Polymer Ed., 6:751-9]. These biocompatible polymers can also resolve discontinuities in the plane of the membrane into an unbroken plasmalemma, and/or become inserted into the membrane defect, sealing it and reversing permeabilization.
For over thirty years polyethylene glycol (PEG) has been known to fuse many cells together to form one giant cell. Application of this hydrophilic macromolecule has been exploited to form multicellular conjugates for the purpose of exchanging genetic material, hybridoma formation, or as a model for endogeneous vesicle fusion [Davidson, R. L., O'Malley, K. A., Wheeler, T. B. (1976): Induction of mammalian somatic cell hybridization by polyethylene glycol, Somat. Cell Genet., 2:271-280; Lee, J., Lentz, B. R. (1997): Evolution of lipid structures during model membrane fusion and the relation of this process to cell membrane fusion, Biochemistry, 36:6251-6259; Lentz, B. R. (1994): Induced membrane fusion; Potential mechanism and relation to cell fusion events, Chem. and Phys. of Lipids, 73: 91-106]. PEG has also been used to fuse many phaetocychroma cells (PC-12; neuron like cells) together to produce large single units facilitating neurophysiological measurements in vitro as well as fusing the severed ends of single invertebrate giant axons in vitro [O'Lague, P. H., Huttner, S. L. (1980): Physiological and morphological studies of rat phechromocytoma calls (PC12) chemically fused and grown in culture, Proc. Nat. Acad. Sci. USA, 77:1701-1705; Krause, T. L., Bittner, G. D. (1990, 1991): Rapid morphological fusion of severed myelinated axons by polyethylene glycol, PNAS, 87: 1471-1475].
Methods and compositions for treating mammalian spinal cord injuries are needed. The present invention addresses these needs.